Project Summary/Abstract Genetic diversity amongst species is due in part to the de novo emergence of new genes from noncoding regions of the genome. New genes that have emerged de novo have been found in every species thus far examined, including hundreds that have been identified in humans, and many disease-associated mutations are found in human-specific genes. ?Proto-genes,? which display features intermediate between genic and non-genic sequences, may represent the pool from which de novo gene birth takes place, but very little is known about the extent, regulation, and physiological consequences of proto-gene expression. The goal of this proposal is to explore proto-gene expression in the budding yeast Saccharomyces cerevisiae through an integration of in silico and in vivo approaches. Proto-gene transcription will be assessed through a re-analysis of RNA-sequencing datasets, which will lead to predictions about the factors that govern proto-gene expression. Proto-gene translation will also be examined by both a re-analysis of mass spectrometry datasets and by high-throughput cloning followed by western blotting, and the structural features of proto-gene translation products will be examined computationally. Finally, by screening a proto-gene overexpression library for effects on fitness under various stress conditions, the adaptive potential of proto- gene expression will be examined. The studies proposed here will enhance our understanding both of the mechanisms that underpin de novo gene birth as well as the cellular function(s) of these proto-genes. !